1. Field of the Invention
This invention relates to the detection of microorganisms. More particularly, it relates to the detection and enumeration of microorganisms in body fluid samples by staining of the microorganisms, subsequent to treatment of the sample with an agent which causes lysis of other components in the sample, so that only the microorganisms are stained. The present invention further relates to a composition, for the above-defined uses, which facilitates simultaneous growth and lysis of components of the sample.
2. Description of the Prior Art
In healthy individuals, most body fluids, such as blood, cerebrospinal fluid or synovial fluid, are sterile, and the presence of microorganisms in these fluids indicates a health problem which, in some cases, may be life threatening. Successful treatment of blood infections requires early diagnosis and proper treatment cannot be initiated until accurate identification of the pathogen has been accomplished. This is made very difficult in the early stages of the infection because the concentration of the pathogen in the blood is very low.
Urine, in contrast, is not a sterile fluid, and microorganisms are present at all times, even in healthy people. Nevertheless, urinary tract infections, generally referred to as bacteriuria, are a major health problem, and there has been considerable debate about what urinary microorganism levels indicate infection and what levels are normal. A review of this subject in light of conventional methods of urinalysis has been presented by Pollock (Am. J. Medicine, Proceedings of a Symposium on Body Fluids and Infectious Diseases, p. 79, July 28, 1983).
For detection of microorganisms in the blood, a variety of systems have been proposed. Two procedures are currently in use in hospital microbiology laboratories. One is a conventional blood culturing method wherein a growth medium is innoculated with the patient's blood and an increase in turbidity, indicative of growth, is monitored over a period of time. The length of time required for growth to result in turbidity is a severe disadvantage. Some organisms require up to 7 days for detection by turbidity.
A second hospital technique is an automated radioisotope detection method wherein the conversion of radioisotope .sup.14 C labeled nutrients to .sup.14 C labeled CO.sub.2 by growth of microorganisms is monitored in the head space gas. This method has been shown to be able to detect 30% of the positive blood cultures in 12 hours and 70% by 24 hours, but is also dependent on growth for detection. Although detection is accomplished sooner than with conventional culturing methods, the 12-24 hour period involved is still a serious drawback where rapid detection of the pathogen is important.
Other methods have been proposed for the rapid detection of septicemia or bacteremia. Some methods have employed various density gradients in an effort to separate the microorganisms from as many blood cell components as possible. Exemplary of these methods is that disclosed in U.S. Pat. No. 4,131,512 to Dorn wherein a lysed blood sample is deposited on a high density liquid cushioning agent and subjected to centrifugation to cause collection of microbial pathogens at the interface between the cushioning agent and the sample. The collected microbial pathogens are then removed from the cushion and cultured on various nutrient agar plates for colony formation.
Current methodology used in the clinical laboratory for the detection of bacteriuria (generally defined as a concentration of microorganisms in the urine of 1.times.10.sup.5 colony forming units per ml (cfu/ml or greater) likewise involves growth-based methods. In these procedures, aliquots of urine (usually 1-10 ul) are cultured for 18-24 hours onto various agar type surfaces and the colonies are counted. These procedures have the disadvantages of the time required for culturing and the cost of materials used on samples which ultimately prove negative.
Staining techniques have also been used for the detection of microorganisms in both blood and urine, and a variety of standard agents such as Gram, Wright, Jenner-Giemsa, Leishman, or May-Grunwald-Giesma stains have been used. For urine samples, these methods have shown a high correlation between uncentrifuged Gram-stained smears and significant bacteriuria. About 90 percent of urine specimens with positive Gram stains have 10.sup.5 cfu/ml or greater. In addition, a high correlation exists between the presence of bacteria on centrifuged wet mounts used in urinalysis, provided the time lag between collection and examination is not overly long. These methods, although good for Gram-positive infections, may not be sufficient for Gram-negative infections, which may be significant at the 10.sup.4 cfu/ml level or in symptomatic patients with 10.sup.2 to 10.sup.4 cfu/ml.
U.S. Pat. No. 4,025,306 to Studer discloses a method whereby microfilariae in the blood are detected by lysis of the blood cells with formaldehyde, staining with a non-fluorescent dye, and observation microscopically. These organisms are the prelarval stage of threadlike worms which invade body cavities and fluids, and are multicelled in contrast to the essentially single celled microorganisms.
Fluorescence microscopy has been used in a variety of inexpensive staining protocols. For example, acridine orange has been utilized to selectively stain bacteria in various clinical samples, including blood and urethral secretions (L. R. McCarthy and J. E. Senne, J. Clin Microb, 11 281 (1980); G. Kronvall and E. Myhre, Acta Path. Microb. Scand 85, 249 (1977). Ethidium bromide has been used extensively to stain both eukaryotic and prokaryotic cells. However, these methods stain not only the microorganisms, but also the white cells and, to a certain extent, other blood components such as platelets and red blood cells. The presence of these other stained bodies makes it difficult not only to locate the microorganisms (which are normally present in small numbers) but also to differentiate between the microorganisms and other stained white cell components and debris.
Accordingly, there is a need for a better method for the rapid detection of microorganisms in body fluid samples. The present invention fulfills this need.